Targeted mRNA Degradation

Targeted mRNA Degradation S Djuranovic and HS Zaher, Washington University, St. Louis, MO, USA r 2016 Elsevier Inc. All rights reserved.

Introduction The central dogma of molecular biology postulates the flow of genetic information from DNA to RNA to protein. This implies that gene expression in cells is a multistep process that involves transcription of genetic material from DNA to RNA molecules, followed by translation of messenger RNAs (mRNAs) into proteins. These processes define normal cell states and they are subject to stringent control at all levels – essentially any step of gene expression can be controlled. Generally, the amount of mRNA in a system is tightly regulated by altering its rate of transcription; however, posttranscriptional control mechanisms fine tune amounts of both mRNA and protein through modification, stability, and degradation of these molecules. Historically, gene regulation has been mainly studied from transcriptional regulation point of view; however, recent genome wide analyses of mRNA and protein abundance in mammalian cells (Rabani et al., 2011; Schwanhausser et al., 2011) revealed that posttranscriptional control plays an equally important part during gene expression regulation. While changes in transcription rates indeed determine the majority of temporal changes in mRNA levels, changes in mRNA degradation rates are found to be important for shaping the dynamic nature of gene regulation which is critical for cell proliferation, cell differentiation, stress, metabolism, immune response, and apoptosis (Ghosh et al., 2010; Houseley and Tollervey, 2009; Schoenberg and Maquat, 2012; Wang et al., 2002). Thus, it comes as no surprise that many cellular factors and mechanisms are employed to specifically regulate and modulate mRNA degradation rates. mRNA stability in eukaryotic cells depends on the presence of a 5′ 7-methylguanosine cap (m7G) and 3′ poly(A) tail, which both serve as integral stability factors for each transcript. These structures also ensure efficient translation of mRNAs through interactions with translation initiation machinery and poly(A)-binding protein (PABP). Loss of these terminal structures from mRNAs through decapping or deadenylation results in their destabilization. mRNAs lacking a m7G cap are rapidly degraded by the exoribonuclease activity of Xrn1 in the 5′-3′ direction. Transcripts lacking a poly(A) tail are eliminated by the 3′-5′ exonucleolytic activity of a large protein complex – exosome. These processes serve as the major pathway by which mRNAs are degraded (Figure 1). We note that initial destabilization by decapping and deadenylation is subject to additional regulation by sequence-specific RNAbinding proteins (RBPs), short RNAs, and by RNA secondary structure, making the process of mRNA degradation specific for each individual transcript. It is worth noting that generation of mRNA through the course of transcription and RNA processing is an imperfect process resulting in a substantial amount of defective transcripts. Furthermore, mRNA is subject to a number of chemical and environmental insults that alter its chemical properties. These mRNAs are subject to quality control processes and

Encyclopedia of Cell Biology, Volume 3

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interestingly their decay appears to be initiated through endonucleolytic cleavage. Since the targets of these processes cause mistranslation, it makes great sense that ‘mRNA surveillance’ depends on the ribosome for the initial recognition of the defect, where the processes are intimately coupled to translation (Shoemaker and Green, 2012).

mRNA Surveillance Mechanisms As discussed earlier, aberrant mRNAs are generated through multiple pathways. The translation of these defective mRNAs is likely to result in aberrant proteins that are likely to misfold and hence cause havoc on the cell. It is not surprising then that cells evolved a number of quality control processes to recognize and degrade aberrant transcripts from the cellular mRNA pool. In eukaryotes, three cytoplasmic quality control processes have so far been identified (Graille and Seraphin, 2012; Kervestin and Jacobson, 2012; Shoemaker and Green, 2012). These are collectively referred to as ‘mRNA surveillance’ and include nonsense-mediated decay (NMD), no-go decay (NGD), and nonstop-decay (NSD) (Figure 2). In NMD, transcripts that contain a premature stop codon (PTC) that could result from mis-splicing, for example, are rapidly degraded. The process of NGD is responsible for degrading transcripts that cause stalling during translation. Finally, NSD acts on mRNAs lacking a stop codon where the ribosome runs to the 3′-end of the mRNA.

Substrate Choice NMD The process of NMD was initially documented in Saccharomyces cerevisiae more than three decades ago, where nonsense mutations in the URA3 gene were found to have profound effects on the stability of its transcript (Losson and Lacroute, 1979). Soon after that, Maquat et al. (1981) showed that β-globin mRNA from β-thalassemia patients, which encodes for a truncated ORF, has a significantly shorter half-life relative to non-thelassemic mRNA. As a result of these initial observations and several others, NMD substrates were classically characterized as transcripts harboring PTCs, which can appear as a result of genomic mutations, transcriptional errors, defective DNA rearrangements, or alternative splicing. The latter process appears to generate many NMD substrates. In particular, it has been estimated that more than 75% of the human pre-mRNA is alternatively spliced and 45% of these produce one mRNA isoform that looks like an NMD target (Lewis et al., 2003). Recent high-throughput transcriptomic analysis, however, suggests that depending on the organism and conditions, 3–15% of the cellular mRNA pool is subject to NMD (Guan

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Figure 1 The main pathways of mRNA degradation in eukaryotes: (a) The canonical degradation pathway. The mRNA is initially deadenylated by the action of a deadenylase complex followed by decapping. The mRNA is then degraded through the action of the 5′–3′ exonuclease Xrn1 and the exosome in the 3′–5′ direction. (b) Degradation of mRNA through endonucleolytic cleavage. The cleavage creates substrates for the exosome, which degrades the 5′ fragment, and Xrn1, which degrades the 3′ fragment.

et al., 2006; He et al., 2003; Lelivelt and Culbertson, 1999; Tani et al., 2012). Furthermore, these NMD targets appear to vary from one cell type to another (Huang et al., 2011; Yepiskoposyan et al., 2011). These findings suggest that NMD, in addition to its role in clearing aberrant mRNA, plays a critical role in gene expression and regulation and appears to be important for a wide range of biological problems. Indeed, whereas NMD is dispensable in some lower eukaryotes such as S. cerevisiae and Caenobharditis elegans, it is absolutely essential in mammals, zebra fish, and fruit fly. In mice, for example, the absence of NMD causes embryonic lethality (Medghalchi et al., 2001). These high-throughput studies and the observation that NMD is involved in a number of biological processes indicate that the classical definition of an NMD target as a PTC-harboring transcript is limited. Emerging from these studies is the discovery that many classes of transcripts can trigger NMD. Transcripts having upstream open reading frames (uORF) are logical targets, where a ribosome translating the uORF encounters that looks like a PTC. 3′-UTRs that have an intron also trigger NMD. Furthermore, long UTR, that are present under normal conditions or result from alternative polyadenylation, can trigger NMD (Chen et al., 2006; Martins et al., 2012), however, not all transcripts with long 3′-UTRs do. Collectively these studies suggest that sequence elements downstream of the stop codon, however, are responsible for trigerring NMD. This unifying theme is often referred to as the ‘faux 3′-UTR’ model (Amrani et al., 2004).

NGD Similar to NMD, NGD was also initially observed in S. cerevisiae; Doma and Parker (2006) showed that mRNAs with strong secondary structures such as hairpins, pseudoknots as well as GC-rich sequences are turned over rapidly. These structures cause translating ribosomes to stall, ‘not go.' While transcripts containing these roadblocks are the most effective NGD targets, the process appears to have other subtle targets that have features that can modulate the speed of the translating ribosome; these include transcripts harboring strings of rare codons or transcripts coding for peptides that, through interactions with the exit tunnel, stall the ribosome (Kuroha et al., 2010; Letzring et al., 2010). It is worth noting that although the use of mRNA reporters with stable secondary structures or long stretches of rare codons have contributed significantly to the dissection of the mechanism of NGD, such mRNAs for the most part do not exist in the transcriptome and hence they have been argued to have limited biological relevance. Recent data suggests that chemically damaged mRNAs, such as oxidized transcripts, are the real targets of NGD. Some of these adducts have been shown to stall the ribosome in vitro (Simms et al., 2014).

NSD As the name suggests, NSD evolved to degrade mRNAs lacking an in-frame stop codon (Frischmeyer et al., 2002; van Hoof et al., 2002). Formally such targets are of two

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Figure 2 mRNA surveillance processes. (a) Canonical termination of protein synthesis. The stop codon is recognized by a ternary complex of eRF1, eRF3, and GTP, which promotes peptide release and initiates recycling of the ribosomal subunits. (b) The exon junction complex (EJC) model of nonsense-mediated decay (NMD). A premature stop codon (PTC) is recognized by eRF1 and eRF3, but due to the presence of an EJC downstream of the stop codon, several other interactions occur with the Upf proteins. These interactions appear to be required for the recruitment of several decay factors. (c) The process of no-go decay (NGD). The yeast eRF1, eRF3, homologues Dom34, and Hbs1 (respectively) recognize the stalled ribosome and bind in the A site. They then catalyze the dissociation of the ribosome and stimulate an endonucleolytic cleavage of the mRNA upstream of the ribosome (not shown). (d) PolyA-mediated nonstop-decay (NSD). In the absence of a stop codon, the ribosome runs to the polyA of the mRNA, synthesizing poly-Lys peptide and effectively stalling the ribosome. The process is similar to NGD in that Dom34 and Hbs1 are sometimes involved but in some organisms other factors are also involved (see text).

different types: the first is generated through truncation or endonucleolytic cleavage and as a result during translation the ribosome simply runs to the end of the message; the second is generated from mutation in the genomic sequence or an error during transcription resulting in mRNAs with no stop codon but with polyA tail. For the latter type, it has been speculated that the ribosome would translate the polyA tail synthesizing long poly-Lys peptide (Ito-Harashima et al., 2007). The polyLys sequence accumulates an overall large positive charge, which is hypothesized to interact with the negative charge of the phosphodiester backbone of the ribosome exit tunnel and induce stalling. Hence, NSD and NGD are similar processes except for where the initial signal for the degradation along the mRNA originates; for NGD it is in the middle, whereas for NSD it is at the end. Indeed, as we shall see later the processes appear to share many of the factors in higher eukaryotes.

Substrate Recognition NMD As mentioned earlier, all NMD targets share the common feature of having a stop codon at a noncanonical position.

Due to the nature of the stop codon translation termination is speculated to be quite distinct from normal termination (Amrani et al., 2004). The exact mechanism by which the ribosome distinguish between a normal stop codon and a PTC is not fully understood, but it involves three conserved proteins: Upf1, Upf2, and Upf3 in S. cerevisiae (Cui et al., 1995; Leeds et al., 1991, 1992). These proteins are thought to bridge a connection between downstream signals and the terminating ribosome (Chakrabarti et al., 2011; Chamieh et al., 2008). The connection between the Upf proteins and the ribosome is thought to be stimulatory for NMD, but inhibitory for termination and they compete with connections that are under normal conditions stimulatory for termination. How do Upf proteins communicate signals to the ribosome? Early studies on NMD suggested that mRNAs harboring a PTC 4B50 nt upstream of exon–exon junction sites triggered a robust NMD response (Nagy and Maquat, 1998; Thermann et al., 1998; Zhang et al., 1998). Later studies showed that during splicing the region just upstream of the exon–exon junction sites is coated with a protein complex fittingly called the exon junction complex (EJC) (Le Hir et al., 2000). During the pioneering round of translation, the EJC complexes are stripped off the mRNA by the ribosome. Given that most of the stop codons reside in the last exon of a gene,

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the presence of an EJC complex downstream of a terminating ribosome provides a rational molecular signature for NMD. The core EJC complex is composed of three proteins, eIF4A3, MLN51, and a MAGOH/Y14 heterodimer, which interacts with the C-terminus of the NMD factor Upf3 (Andersen et al., 2006; Bono et al., 2006; Buchwald et al., 2010). Upf3 has orthologoues in all examined eukaryotes and consistent with its interaction with the EJC complex it shuttles between the nucleus and cytoplasm (Kim et al., 2001). Upf2 acts as a bridge between Upf3 and Upf1, which in turn interacts with the release factors eRF1 and eRF3 as well as the phosphatidylinositol 3-kinase-related kinase SMG1 (Kashima et al., 2006; Kunz et al., 2006). Among NMD factors, Upf1 is the most conserved factor and is central to the process (Culbertson and Leeds, 2003). Sequence analysis as well as biochemical characterization of the protein revealed that it belongs to the ATPdependent helicase superfamily (Cheng et al., 2007; Czaplinski et al., 1995). It binds RNA and ATP and uses the energy from ATP hydrolysis to unwind RNA in a 5′ to 3′ direction. In addition, the protein is subject to cycles of phosphorylation (by SMG1) and dephosphorylation on its unstructured C-terminus and N-terminus region (Grimson et al., 2004; OkadaKatsuhata et al., 2012; Yamashita et al., 2001). On NMD substrates, Upf1–Upf2–Upf3 form a stable complex and it has been suggested that interaction between Upf2 and a cysteine/ histidine (CH) rich domain on Upf1 activates the ATPase and helicase activity of Upf1 (Chamieh et al., 2008). In the absence of Upf2, the CH domain of Upf1 interacts with the factor’s helicase domain forming a closed conformation that inhibits the unwinding activity of Upf1. In addition to their interaction with the EJC complex, Upf proteins have been shown to interact with translation factors, namely release factors, for which they appear to modulate termination efficiency (Kashima et al., 2006). Stop codon readthrough is significantly more efficient on PTCs relative to normal stop codons (Wang et al., 2001). What is clear from the many decades of studies on NMD is that the process is intimately coupled to translation termination. Termination of protein synthesis occurs when one of three nearly universal stop codons (UAG, UGA, and UAA) enter the A site of the ribosome. In eukaryotes, termination requires the two release factors eRF1 and eRF3. eRF1 is a tRNA mimic, which binds the A site of the ribosome and recognizes the stop codon. eRF3 is a GTPase and similar to EF1A (Frolova et al., 1996), which binds aa-tRNAs to deliver them to the ribosome during elongation, is thought to form a ternary complex with eRF1 and GTP (Mitkevich et al., 2006). Once on a terminating ribosome, GTP is hydrolyzed, eRF1 undergoes a conformational change allowing the conserved GGQ domain to engage the active site of the ribosome to initiate peptide release. It has also been suggested that eRF3 interacts with polyA-binding protein (Pab1), and that this interaction stimulates peptide release (Kononenko et al., 2010). Following peptide release, the conserved recycling factor ABCE1 binds to eRF1 kicking eRF3 off; subunit dissociation ensues to complete the translation cycle (Pisarev et al., 2010). Based on this relatively short description of termination, it should be apparent that the process is amenable to modulation through differential protein–protein interaction network. Indeed on NMD substrates, as detailed earlier, termination is inhibited due to

what has been suggested as direct competition between the Upf proteins and termination-stimulating factors (Ivanov et al., 2008). In particular, Upf1 has been shown to interact with eRF3, but whether this interaction on its own is sufficient to elicit NMD is debated. While the EJC model is elegant and appealing, it fails to explain all of the targets of NMD. An increasing number of NMD-targeted mRNAs do not have an intron downstream of a stop codon and as such are predicted not to harbor an EJC complex. Furthermore, NMD is robust in yeast, even though most of the pre-mRNAs are not spliced. It is worth noting that immunoprecipitation of eIF4AIII (a core component of the EJC) followed by deep sequencing suggests that the EJC can be deposited on mRNAs in a sequence-dependent and splicingindependent manner (Sauliere et al., 2012; Singh et al., 2012). So the possibility that the EJC is found on the UTR of mRNAs lacking an intron downstream of the stop codon cannot be ruled out. Furthermore, for all of these noncanonical targets, the mRNAs share the common feature of having a long UTR. The UTR model suggests that Upf1 binds to mRNAs nonspecifically and during translation the ribosome actively displaces the factor (Hogg and Goff, 2010). Since the ribosome does not traverse the 3′-UTR, mRNAs with long UTR are expected to have a larger number of Upf1 proteins and hence are subject to NMD. Alternatively, it has been suggested that the polyA tail of mRNAs with long 3′-UTRs is distant from the stop codon, precluding any stimulatory effect PAB1 might have on termination. In contrast, others have argued that in the absence of a downstream EJC complex Upf2 and Upf3 interact with a ribosome-bound Upf1, which on its own is able to sense the efficiency of termination (Stalder and Muehlemann, 2008). In summary, Upf1 appears to be the primary factor in determining what constitutes an NMD target whatever the initiating signal might be. Future work is needed to clarify the mechanism of the initial recognition of a PTC.

NGD NGD is triggered by a stalled ribosome and requires the conserved protein Dom34 (or Pelota) and Hbs1. Interestingly, Dom34 and Hbs1 share structural features with eRF1 and eRF3, respectively (Atkinson et al., 2008; Chen et al., 2010). Consistent with these structural similarities, the factors interact with the A site of the ribosome (Becker et al., 2011) but because Dom34 lacks the GGQ domain of eRF1, the complex does not promote peptide release. Instead, in vitro biochemical studies showed that the factors promote subunit dissociation (Shoemaker et al., 2010). Moreover, the recycling factor ABCE1 (Rli1 in yeast) plays an important role during this reaction (Shoemaker and Green, 2011). How does Dom34–Hbs1 complex recognize a stalled ribosome? How does it compete effectively with the ternary complex of EF1–aa-tRNA–GTP that normally binds the A site during elongation? Important clues into this process emerged from recent biochemical characterization showing an inverse correlation between the efficiency of subunit dissociation activity and the length of the mRNA downstream of the P site (Pisareva et al., 2011; Shoemaker and Green, 2011). In particular, Dom34–Hbs1 appears to prefer ribosomal complexes

Cell Division/Death: Regulation of Cell Growth: Targeted mRNA Degradation

with little sequence downstream of the P site. Later structural studies showed that this length dependency is bestowed by Hbs1. The factor binds the ribosome in the mRNA entry tunnel and hence can only bind when the mRNA is short (Becker et al., 2011).

NSD As mentioned earlier, NSD is similar to NGD except that the former involves ribosomes stalling at the end of the mRNA. As a result, the processes proceed in a similar fashion utilizing the same factors. A notable exception is the requirement of Ski7 for NSD in some species of yeast (van Hoof, 2005). The factor is a translational GTPase related to eRF3 and Hbs1. However, in contrast to eRF3 and Hbs1, which have binding partners (eRF1 and Dom34, respectively), no binding partner for Ski7 has been identified. As a result, the mechanism of the recognition of NSD targets in yeast remains poorly understood. It is worth noting that Ski7 interacts with the exosome, thereby linking ribosome recognition of NSD targets to their ultimate degradation (van Hoof et al., 2002).

Substrate Fate The ultimate fate of the aberrant mRNA, regardless of the process (NMD, NGD, or NSD), is degradation. All of the pathways eventually utilize canonical decay pathways in both directions: 5′–3′ Xrn1-mediated and 3′–5′ exosome-mediated. Decay of metazoan NMD targets appears to initiate with an endonucleolytic cleavage catalyzed by Smg6 (Eberle et al., 2009; Gatfield and Izaurralde, 2004). Following this cleavage, the 5′ and 3′ pieces are degraded by the exosome and Xrn1, respectively. Nonetheless, accumulating evidence suggests that alternative and potentially redundant mechanisms exist for clearing NMD targets that do not include endonucleolytic cleavage. For instance, yeast does not have a Smg6 homologue and no cleavage to date has been observed on NMD targets, yet decay is robust in this organism. Furthermore, tethering of the NMD factor Smg7 to mRNAs, in the absence of the endonuclease Smg6, results in fast degradation of the mRNA (Unterholzner and Izaurralde, 2004). Therefore, it has been suggested that decay of NMD targets can also proceed through exonucleolytic routes. The Smg6–Smg7 complex has been shown to recruit decay factors, mainly the deadenylation machinery, to accelerate degradation of NMD targets (Mitchell and Tollervey, 2003; Muhlrad and Parker, 1994). NSD and NGD targets are subject to endonucleolytic cleavage. The cleavage has been shown to occur upstream of the stalled ribosome. For NSD, the catalytic subunit of the exosome rrp44 (Dis3), which has endonucleolytic and exonucleolytic activities, has been implicated in catalyzing the initial cleavage reaction (Schaeffer et al., 2009; Schaeffer and van Hoof, 2011). For NGD, the endonuclease is yet to be identified, but Dom34–Hbs1 (although unnecessary for the cleavage activity) appears to stimulate the reaction (Tsuboi et al., 2012). In summary, the three mRNA surveillance processes to a large extent exploit similar mechanisms to initiate the decay of their targets. There is a lot to learn about the

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process of recognition of the targets and the mechanism of preparing them for degradation.

Targeted mRNA Degradation in Gene Regulation In the first part we focused on the process of mRNA degradation due to interruptions in translation cycle that ultimately leads to activation of mRNA surveillance mechanisms. However, as we mentioned earlier, each mRNA transcript will have different set of bound RBPs and RNPs and differences in cellular presence of such trans regulatory elements will lead toward variation in mRNA degradation rates and differential gene regulation. The changes in mRNA degradation rates due to the targeted binding of trans elements as a subject of gene regulation control are described in nearly all cellular pathways from development to oncogenesis (Keene, 2007; Kishore et al., 2010). As such eukaryotic mRNA degradation rates can vary remarkably from transcript to transcript as well as for the same transcript from cell to cell. Cellular modulators of mRNA degradation rates are mainly short RNAs and RBPs. The principle underlying the modulation of mRNA degradation rates by mRNPs involves controlled exposure of mRNA molecules to cellular RNA degradation machineries. This requires either removal of ‘stabilizing’ RBPs attached to mRNA transcripts and/or recruitment of ‘destabilizing’ RBPs and short RNAs. The vulnerable ‘exposure’ of mRNAs to short RNAs and RBPs dictate particular mRNA decay patterns of such mRNPs via direct or indirect interactions with the enzymes controlling mRNA degradation, mRNA deadenylation, or decapping factors (Figure 3).

mRNA Degradation Control by Short RNAs microRNA (miRNAs) are short (about 21–25 nt long) noncoding RNAs that regulate the translation and degradation rates of mRNAs through nearly perfect complementary binding of target sequences in mRNA 3′ untranslated regions (UTRs) (Bartel, 2004). While originally discovered in the nematode C. elegans (Lee et al., 1993; Wightman et al., 1993), they are now regarded as one of the key regulators of genes in most metazoans (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). miRNAs are thought to regulate the expression of target genes by translational repression and targeted mRNA degradation (Bazzini et al., 2012; Bethune et al., 2012; Djuranovic et al., 2012; Eulalio et al., 2009; Guo et al., 2010; Meijer et al., 2013; Figure 3). These two processes are coupled in the living cell and whether the regulatory effects of miRNAs on target genes are the result of the direct induction of mRNA degradation or effects on mRNA decay are the result of an early block in translation initiation, consequentially leading to rapid mRNA degradation still remains elusive (Djuranovic et al., 2011; Fabian et al., 2010). In both cases miRNAs end up MukMuenhancing mRNA target degradation by directing mRNAs to the regular exonucleolytic mRNA decay involving initial deadenylation of the mRNA via the CCR4–POP2–NOT complex (Huntzinger and Izaurralde, 2011; Figure 3). Effects of miRNAs on mRNA target occur through ribonucleoprotein complexes named miRISCs, which at minimum include the

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Figure 3 The basic mechanisms controlling mRNA degradation by microRNA (miRNAs) and RNA-binding proteins (RBPs). Recruitment of the mRNA regulators, such as miRNAs or RBPs, triggers either translational repression or direct destabilization of target mRNAs ultimately leading toward mRNA degradation. RBPs and miRISC (miRNA–AGO–GW182 complex) act directly on translation through interaction with translation initiation factors (eIF3, eIF4A, eIF4E, eIF4G) or PABP, preventing active translation of targeted mRNA and exposing it to mRNA degradation machinery. In alternative pathway, RNA regulators directly recruit mRNA destabilization factors, involved in decapping and deadenylation. The removal of the protective structures from either 5′ (m7Gppp-cap) or 3′-terminus (poly(A)-tail) of target mRNA results in RNA degradation by Xrn1 (5′–3′ decay) and the exosome (3′–5′ decay).

miRNA, Argonaute (AGO), and GW182 protein (Wilson and Doudna, 2013). The binding of such minimal miRISC complex is sufficient for translational repression which in turn might be sufficient to make mRNA targets ‘vulnerable’ to mRNA degradation machinery (Djuranovic et al., 2011). Additionally it was also shown that direct interactions of AGO or GW182 proteins with both deadenylation and decapping factors generate more substantial effects on mRNA degradation (Braun et al., 2011). An interesting fact is that targeting of mRNAs by miRNAs often leads to localization of target mRNAs in cytoplasmic foci termed P-bodies which can act as a site for both storage and degradation of target mRNAs (Parker and Sheth, 2007). It is plausible to think that the complexity of miRNA biogenesis (Ha and Kim, 2014), mechanisms of interaction between miRNAs and target mRNAs (Bartel, 2009) as well as existence of different miRISC complexes (Wu et al., 2013) offer numerous possibilities for modulation of miRNA mediated gene regulation and targeted mRNA degradation.

More recently it was shown that piRNAs, another class of short RNAs, may control mRNA degradation patterns in multiple organisms (Gou et al., 2014; Kiuchi et al., 2014; Rouget et al., 2010; Watanabe et al., 2015). piRNAs are known for their function in the repression of transposable elements primarily within the germ line cells, however their function might be also important in regulation of gene expression (Thomson and Lin, 2009). piRNAs in early Drosophila embryos affect nanos mRNA deadenylation and degradation through the interplay with both Smaug, an RNA-binding protein, and already mentioned CCR4–POP2–NOT deadenylation complex (Rouget et al., 2010). In a similar manner, interactions of piRNA-silencing complex (piRISC), containing piRNAs and murine PIWI protein (MIWI), and CCR4–CAF1–NOT deadenylase complex in mouse testes controls expression of approximately 5000 genes through targeted mRNA destabilization through CAF1-dependent deadenylation (Gou et al., 2014).

Cell Division/Death: Regulation of Cell Growth: Targeted mRNA Degradation

Targeted mRNA Degradation by RBPs The main feature of RBPs is the presence of at least one RNAbinding domain in their protein sequence (Lunde et al., 2007). One of the best characterized families of RBPs linked to mRNA degradation is the Hu, ELAV proteins (Brennan and Steitz, 2001; Simone and Keene, 2013). This group of proteins specifically interacts with 3′ UTRs of mRNA transcripts containing adenylate/uridylate-rich elements (AREs), cis-regulatory sequences linked to mRNA degradation control (Shaw and Kamen, 1986). The addition of ARE elements derived from the human colony stimulating factor 2 (CSF2) gene to the 3′ UTR sequences of a stable reporter mRNA, such as β-globin, results in an unstable chimeric transcript. Such experiments demonstrated the importance of these sequence elements in the stability of mRNAs in the cell (Chen and Shyu, 1995; Shaw and Kamen, 1986). The importance of Hu/ELAV proteins and their biological significance is especially highlighted by their function in neurons. Pathological conditions affecting the level of functional Hu/ELAV proteins lead to severe brain damage pathologies that are quite often connected to degenerative neurological disorders following paraneoplastic syndromes (Darnell, 2013; Pascale et al., 2008). Bioinformatic analyses estimated that 5–10% of the human transcriptome contain AREs and appear to be essential in regulating apoptosis, immune response, and intracellular signaling to name a few (Halees et al., 2008). The exact mechanism by which Hu/Elav and other ARE binding proteins lead to a change in mRNA stability are not yet fully resolved. In one particular example, HuR positively regulates the stability of the mRNA transcript of the human eukaryotic translation initiation factor 4E (eIF4E). The process involves direct competition between HuR and yet another ARE binding factor – AUF1 (Topisirovic et al., 2009). AUF1 has been implicated in RNA decay regulation through its binding to eukaryotic initiation factor 4G (eIF4G) and PABP ultimately leading to mRNA destabilization (Lu et al., 2006). Mechanistically, molecular interaction between AUF1, eIF4G, and PABP may lead to the interruption in translation cycle or/ and displacement of PABP from poly(A) tail of AUF1 bound transcripts allowing the deadenylase complex access to the mRNA (Sagliocco et al., 2006). Additionally, AUF1 may facilitate degradation of targeted mRNAs by direct recruiting of exosomes to ARE–mRNA complexes (Chen et al., 2001; Torrisani et al., 2007). At a molecular level, by binding to AREs, AUF1 or HuR are likely to alter some local RNA structure, which in turn provides necessary surface area for additional RBPs or RNPs and hence alters mRNA translation and stability (Wilson et al., 2003; Zucconi et al., 2010). Given that majority (57%) of HuR targets are also bound by AUF1, antagonizing effects and competitive binding to AREs between these two RBPs might be relevant for the stability of many mRNA targets (Lal et al., 2004). In addition to already mentioned HuR/ELAV and AUF1, several other RBPs are also known to interact with AU-rich elements. These among others include – tristetraprolin (TTP) (Lai et al., 1999), butyrate response factor-1 (BRF1) (Stoecklin et al., 2002) and KH-type splicing regulatory protein (KSRP) (Gherzi et al., 2004). The KH domains of KSRP are essential for their degradation activity via interaction with deadenylase PARN, decapping factor Dcp2, and exosome subunit Rrp4 (Chou et al., 2006; Gherzi et al., 2004). TTP and

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BRF1 interact specifically with exosomes, the deadenylase complex (involving Ccr4), decapping enzymes, and Xrn1 to promote mRNA degradation in P-bodies through a sequential process (Fenger-Gron et al., 2005; Franks and Lykke-Andersen, 2007; Lykke-Andersen and Wagner, 2005). Other RBP such as Smaug are also important in controlling translation and degradation of targeted mRNAs. Smaug belongs to a family of conserved sterile alpha motif domain containing RBP and all members of this family are thought to be involved in posttranscriptional gene regulation (Aviv et al., 2003). In Drosophila, Smaug plays an important role in posttranscriptional regulation of the RNA-binding protein Nanos (Nos) (Chen et al., 2014). Smaug carries out this function by binding Smaug recognition elements (SREs) in the Nos 3′UTR, as well as in other 3′ UTRs. This feature is seen across many RBPs since the specificity for target interaction depends on both primary sequence and secondary structure features (Smibert et al., 1996). Beside the effect on translation inhibition, Smaug was later shown to control the stability of targeted mRNAs by recruiting the deadenylase complex CCR4– POP2–NOT (Semotok et al., 2005). In addition to those mentioned above, several other cisregulatory elements have been related to RNA degradation control. These include U-rich, CU-rich, GC-rich, poly(C), CArich, and GU-rich (GRE) elements (Hamilton et al., 2008; Rattenbacher et al., 2010; Stoecklin et al., 2002; You et al., 1992), which are regulated by a wide range of RBPs. Moreover, with a recent delineation of an ‘atlas of mammalian mRNA binding proteins’ (Castello et al., 2012) which identified more than 300 newly described RBPs and emerging variety of cisregulatory mRNA degradation elements the complex regulatory landscape controlling the fate of mRNA molecules in the cell is still far from being completely explored.

Combined Action of Short RNAs and RBPs Last but not least, it is plausible that dynamic combination of miRNAs and RBPs that are associated to most mRNAs modulates these two types of RNA regulatory interactions and that the outcome of such interactions affects the mRNA degradation patterns in yet completely novel fashion (Figure 3). It was shown that binding of HuR protein to the 3′ UTR of the CAT1 mRNA in human cells attenuates the repressive effects of miR-122 on CAT1 (Bhattacharyya et al., 2006). These experiments were further confirmed using reporter mRNA targets as well as in vitro experiments (Kundu et al., 2012) showing that binding of Hur to mRNA transcripts targeted by miRNAs ultimately leads to the dissociation of miRISC from mRNA target. Furthermore, HuR association with miRNA targets can also inhibit miRNA-induced mRNA deadenylation and degradation. The possible mechanistic explanation for antagonizing actions of HuR protein on miRISC targets came from more recent study on another ARE binding protein HuD (Fukao et al., 2014). While miRISC promoted efficient release of eukaryotic initiation factor 4A (eIF4A) from target mRNAs (Fukao et al., 2014) causing translational repression, HuD protein counteracted this miRISC action by stabilizing binding of eIF4A protein to target mRNAs ensuring efficient translation of such transcripts. In some other cases, RBPs can actually

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enhance miRNA regulation. For instance, in mammalian cells, the Pumilio RBPs Pum1 and Pum2 promote the regulatory effects of miR-221/miR-222 on the p27 mRNA (Kedde et al., 2010). In regulation of p27 by Pum1 and miR-221/222, Pum1 binds to a stem-loop containing Pumilio recognition element (PRE) and the miR-221/222 target site relaxing the RNA secondary structure and allowing miRNA binding. These interactions are significant, given that p27 downregulation by miR221/222 is essential for cell proliferation and may also have a central role in the development of cancer (Triboulet and Gregory, 2010). In the case of E2F mRNA it was demonstrated that removal of PREs or depletion of Pum1/2 prevented miRNA regulation of the E2F mRNA. It is currently unknown how Pum1/2 in conjunction with miRNAs regulates E2F transcript translation and stability (Miles et al., 2012). It is possible that binding of Pum1/2 induces RNA structural rearrangements, however these changes would need to affect three different miRNA binding sites over a several hundred base-pair long distance. At the end, these examples are here illustrating the importance of the physical interactions between mRNA, RBPs, and miRICS to achieve efficient gene regulation through the modulation of RNA degradation rates.

See also: Nucleic Acid Synthesis/Breakdown: RNA Synthesis/ Function: Messenger RNA (mRNA): The Link between DNA and Protein

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